CN103389237A - Simple low-cost microarray chip sample applicator and application method thereof - Google Patents
Simple low-cost microarray chip sample applicator and application method thereof Download PDFInfo
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- CN103389237A CN103389237A CN2013103301434A CN201310330143A CN103389237A CN 103389237 A CN103389237 A CN 103389237A CN 2013103301434 A CN2013103301434 A CN 2013103301434A CN 201310330143 A CN201310330143 A CN 201310330143A CN 103389237 A CN103389237 A CN 103389237A
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Abstract
The invention discloses a simple low-cost microarray chip sample applicator and an application method thereof. The sample applicator is composed of a silicone rubber chip comprising a micro-through-hole array and a group of micro-pipelines, wherein each micro-through-hole arranged on the chip is communicated with at least one micro-pipeline, each micro-pipeline is independent, and each micro-pipeline is connected with at least one injection port. The application method comprises the following steps of firstly placing the sample applicator in a vacuum environment so as to carry out degasification treatment, fitting the sample applicator after degasification treatment with a substrate to be subjected to sample application, contacting one surface of the sample applicator which contains the micro-through-hole array with the substrate to be subjected to sample application, dripping the corresponding sample to be fixed on each injection port, absorbing negative pressure formed by air in the micro-pipelines by utilizing a degasification silicone rubber block so as to drive a liquid sample of the injection ports to fill with the micro-pipelines and the micro-through-hole array, standing for a certain time, finishing a cross-linking reaction on the liquid sample in the micro-through-hole array and the surface of the substrate, peeling the sample applicator, and cleaning the substrate, thereby obtaining the micro-array chip which finishes sample application.
Description
Technical field
The present invention relates to a kind of micro-array chip sample applicator and using method, described micro-array chip sample applicator has simple and easy and low-cost characteristics or rather, and described sample applicator can be applicable to the fields such as biomedical high throughput analysis and screening.
Background technology
Micro-array chip (Microarray) is a kind of new molecular biology research instrument that developed recently gets up, it is by arranging in an orderly manner molecule or the biological sample that has in a large number bio-identification function at substrate surface, utilize special interaction between biomolecule, realize the high flux fast detecting to nucleic acid, polypeptide, protein and other biological composition.The micro-array chip technology can be at Multiple components or the virulence factor of specificity in detecting sample in experiment once; all be with a wide range of applications in a plurality of fields such as gene studies, clinical diagnosis, new drug development, environmental protection, become one of revolutionary cutting edge technology of current biomedical sector.Although the micro-array chip technology has very outstanding technical advantage, though also experienced the development of recent two decades, present and fail to acquire widespread use, main cause is that the micro-array chip price is too high, has limited its userbase.So, realize the widespread use of micro-array chip technology, key is to reduce the application cost of micro-array chip.The micro-array chip manufacturing technology mainly comprises at present: contact point sample technology and contactless point sample technology; Contact point sample technology is generally to utilize the point sample syringe needle directly to contact with substrate surface, realizes the orderly distribution of sample at substrate surface; And contactless point sample technology type is similar to inkjet technology, utilizes the methods such as piezoelectricity, static or thermal expansion that the sample in mini sprinkler is injected into substrate surface, realizes the micro-array chip making.Although these two kinds of technology are all quite ripe, but many Related products are arranged also on market, but all there is following limitation in actual applications in these two kinds of technology: 1. point sample equipment point sample syringe needle used (or shower nozzle) and spotting robot are expensive, cause the micro-array chip cost of manufacture high; 2. point sample equipment is accurate complicated, needs the technician's operation through special training, has increased human cost; 3. the point sample equipment volume is huge, and environmental baseline is had higher requirements, and needs special parking space; 4. point sample equipment point sample syringe needle used (or shower nozzle) is and reuses, and has the danger of sample cross pollution; 5. the point sample process is responsive to character such as the surface tension of sample and viscosity, easily cause different samples in the heterogeneity of final point sample effect and inconsistent, thereby affect reliability and confidence level [the Barbulovic-Nad I of data analysis, Lucente M, Sun Y, Zhang M, Wheeler AR, Bussmann M.Bio-microarray fabrication techniques--a review.Crit Rev Biotechnol.2006,26 (4): 237-59; Dufva M.Fabrication of high quality microarrays.Biomol Eng.2005,22 (5-6): 173-84.].In addition, although these two kinds of technology are in chip manufacturing in enormous quantities, advantage with robotization and speed aspect, still be not suitable for short run, DIY(Do it yourself for micro-array chip) make, therefore be difficult to be widely applied in numerous common lab.In order to realize the widespread use of micro-array chip technology, the micro-array chip user scope is expanded to hospital and the common lab of One's name is legion from large-scale R﹠D institution, real performance microarray technology is in the advantage in the fields such as clinical diagnosis, gene studies, new drug development, in the urgent need to developing a kind of novel, low-cost, easy micro-array chip preparation facilities, cost of manufacture with the decrease micro-array chip, expanding its application scale, is directly hundreds of millions patients and numerous medium and small laboratory service.This becomes design of the present invention undoubtedly.
Summary of the invention
The object of the present invention is to provide a kind of Simple low-cost micro-array chip sample applicator, the sample applicator that provides need not accurate expensive mechanical hook-up and external supply unit, can easy, promptly prepare high-quality micro-array chip, have characteristics easy and simple to handle, with low cost, thus be expected to realize the micro-array chip technology aspect clinical and high flux biochemical analysis popularization and application.
A kind of Simple low-cost micro-array chip sample applicator provided by the invention, it is characterized in that: described sample applicator is a silastic chip that comprises micro through hole array and one group of microchannel, on chip, each through hole of micro through hole array communicates with a microchannel at least, separate between each microchannel, and every microchannel connects an injection port at least.Described sample applicator comprises at least one micro through hole array, and all via hole geometry yardsticks of each micro through hole array are all consistent, the geometric configuration of through hole can in circular, ellipse and polygon any one.Arrange for simple sample dot matrix, each of sample applicator microchannel can be positioned at same layer; Dot matrix for complexity is arranged, and in order to prevent the intersection of sample mixing in each pipeline in the sample assigning process, each of sample applicator microchannel can be positioned at two layers and above different layers, and the microchannel of different layers all is connected with micro through hole with corresponding injection port.
According to the effect in use procedure, described sample applicator comprises up and down two function surfaces (as shown in Figure 2), and upper surface comprises injection port, is used for application of sample; Lower surface comprises micro through hole array, is used for restriction and treats fixed sample and substrate contact reaction zone.And all the other are flat surface to the sample applicator lower surface except the micro through hole zone, to guarantee fitting tightly of point sample process mid-point sample device and substrate; The lower surface area should be equal to or less than treats the point sample chip area, to guarantee that in the point sample process, all micro through hole array all are seated on substrate.
The using method of micro-array chip sample applicator of the present invention is as follows: at first, sample applicator is placed in vacuum tank, and (pressure＜10kPa) carries out degassed processing at least 40 minutes, sample applicator after degassed processing can directly be applied after taking out from vacuum tank, and is also salable standby at any time in vacuum bag; Sample applicator after degassed processing takes out from vacuum tank or vacuum seal bag, with treat the point sample substrate and aim at laminating, wherein sample applicator lower surface (one side that namely comprises micro through hole array) contacts substrate, because silicon rubber has good compliance, can be attached at well smooth glass surface by van der Waals interaction, realize perfect sealing; After completing the laminating assembling, drip and treat fixed sample solution at each injection port of sample applicator upper surface, make sample solution seal each microchannel injection port, form the microchannel closed system, because the silicon rubber block after degassed processing has higher dissolved gas characteristic, so the air in the sample applicator microchannel can continue to be made in sealing microchannel system and produce negative pressure by its silicon rubber tube wall absorption, form the fluid driving force, drive fluid sample and be full of microchannel and micro through hole array; After sample liquids is full of sample applicator microchannel and microarray, seal each injection port with oil droplet, to prevent the sample liquid moisture evaporation, and the substrate that will assemble sample applicator is placed in suitable temperature and humidity environment, standing more than at least 30 minutes, peel off sample applicator from substrate finally, and substrate is immersed in suitable cleaning fluid and cleans, remove unreacted sample molecule, dry up.Because the fluid sample of filling in sample applicator only contacts with substrate surface in through hole, cross-linking reaction occurs, therefore after finally peeling off sample applicator, cleaning unreacted sample molecule, substrate surface is only forming sample spot with sample applicator through hole contact position, be that substrate surface forms the sample spot microarray corresponding with the sample applicator micro through hole array, make the micro-array chip based on the definition of sample applicator micro through hole array.
For traditional pin type contact point sample technology and non-contacting ink jet type point sample technology, in the point sample process, be dispensed to the little drop of each sample of substrate surface, except bottom surface is subjected to the substrate restriction, all the other all directions all are in open state, therefore can flow freely, can affect the contact area (sample secure bond area) of drop and substrate when the appearance such as the surface energy variation of volume, density, surface tension and the substrate of drop and difference; In addition, pin type contact point sample technology realizes sampling based on capillary action, and its sample volume also is subjected to the sample liquid effect of surface tension, and the dosing of ink jet type point sample technical point is dripped in process, and the liquid droplets size also is subjected to the impact of sample liquid surface tension and viscosity.Therefore, for traditional pin type contact point sample technology and non-contacting ink jet type point sample, impact due to the factors such as instability of the difference of the character such as sample liquid surface tension, viscosity and substrate surface character, cause being difficult to making and have good homogeneity and conforming micro-array chip, thereby limited the application of micro-array chip technology.And point sample method provided by the invention makes the contact area (being the secure bond zone) of sample liquid and substrate limited by the micro through hole opening, therefore the shape of the sample spot that forms is only relevant to the micro through hole opening with size, and not affected by the factors such as sample liquid surface tension, viscosity, density and substrate surface energy, therefore utilize this technology only need make the micro through hole array of homogeneous, can prepare easily and have good homogeneity and conforming micro-array chip.
Micro-array chip based on silastic chip sample applicator provided by the invention, compare with the existing spot sample device of microarray based on mechanical arm, need not accurate expensive mechanical hook-up and the operating personnel of specialty, also need not external energy supply equipment and huge placement space, but the cost of manufacture of decrease micro-array chip, and this sample applicator has higher dirigibility, be applicable to common lab and carry out short run and the DIY preparation of micro-array chip, therefore be expected to realize the popularization and application of micro-array chip technology; Simultaneously, the point sample effect of this sample applicator is not treated the impact of the factors such as the surface tension, viscosity of point sample liquid, has guaranteed the consistance that micro-array chip is made, and can disposablely use, avoid cross pollution, can greatly improve the reliability of micro-array chip technology application.
Sample applicator of the present invention need not accurate expensive mechanical hook-up, also need not the operating personnel of external energy supply equipment and specialty, but the cost of manufacture of decrease micro-array chip realizes the popularization and application of micro-array chip technology; Simultaneously, the point sample effect of this sample applicator is not treated the impact of the character such as the surface tension of liquid, viscosity of point sample, can greatly improve consistance and reliability that micro-array chip is made.
Description of drawings
Fig. 1 is the sample applicator structural representation that the present invention is based on the micro through hole array of individual layer microchannel connection, in figure: (a) sample applicator, (b) be the A profile, be (c) the B profile, be (d) C section partial enlarged drawing.
Fig. 2 is sample applicator surface structure schematic diagram shown in Figure 1 (figure (a)) and lower surface configuration schematic diagram (figure (b)).
Fig. 3 is that sample applicator shown in Figure 1 uses flow process, in figure: (a) the degassed processing of sample applicator, (b) degassed rear sample applicator and slide laminating, (c) injection port liquid filling, (d) complete micro-array chip and make.
Fig. 4 is the sample applicator structural representation that the present invention is based on the micro through hole array of multilayer microchannel connection.。
Embodiment
Further illustrate substantive distinguishing features of the present invention and significant progressive below in conjunction with embodiment.
As shown in Figure 1, simple and easy, low-cost micro-array chip sample applicator provided by the invention, it is characterized in that described sample applicator (1) is a silastic chip that comprises micro through hole array and one group of microchannel, on chip, each through hole (2) of micro through hole array communicates with a microchannel (3) at least, separate between each microchannel, and every microchannel connects an injection port (4) at least.And for example to comprise the sample applicator structural representation (a) of upper and lower two function surfaces be upper surface to Fig. 2, (b) is lower surface.
at first (pressure＜10kPa) carries out degassed processing (as shown in Fig. 3 a) sample applicator (1) to be placed in vacuum tank (5), after degassed 1 hour, take out sample applicator, and it is comprised the one side of micro through hole array and aldehyde group modified slide (6) laminating (as shown in Fig. 3 b), then drip respectively 1 μ L different antibodies solution (7) at each injection port, absorb at degassed silicone rubber under the effect of microchannel Air formation negative pressure, injection port liquid filling microchannel and micro through hole array (as shown in Figure 3 c), after all antibody-solutions are full of microchannel and micro through hole array, drip 2 μ L paraffin oils at each injection port, and with the assembly of sample applicator and slide, be positioned in 25 ℃ of environment, standing 24 hours.Finally, from slide, peel off sample applicator, and slide is immersed in cleaning fluid and shakes 10min, with rinsed with deionized water, nitrogen dries up, and completes the preparation (as shown in Fig. 3 d) of Antibody microarray chip (8).
For the microarray point sample figure of complexity, above-mentioned sample applicator based on the individual layer microchannel often is difficult to realize because each microchannel is connected with micro through hole array arrange in unavoidably appearance mutually intersect.In order to keep separate between each microchannel, prevent the mixing of crosstalking of mutual sample that the intersection of each microchannel causes, realize complicated microarray point sample figure, can adopt the multilayer microtube structure to build, during individual layer is arranged, cross one another microchannel is distributed in different structural sheets.Such as contain 36 point sample sites for each microarray, and require the point sample of the fixing different samples in each point sample site in single microarray to require, the sample applicator that can adopt three-decker as shown in Figure 4 to build is realized, this sample applicator consists of via layer (9), the first pipe layers (10) and second pipe layer (11), Fig. 4 left side structure (a) is exploded perspective view, is (b) assembly drawing.
Claims (8)
1. Simple low-cost micro-array chip sample applicator, it is characterized in that described sample applicator is a silastic chip that comprises micro through hole array and one group of microchannel, on chip, each through hole of micro through hole array communicates with a microchannel at least, separate between each microchannel, and every microchannel connects an injection port at least.
2. sample applicator according to claim 1, it is characterized in that described sample applicator comprises at least one micro through hole array, and all via hole geometry yardsticks of each micro through hole array are all consistent, shape of through holes can in circular, ellipse and polygon any one.
3. sample applicator according to claim 1, is characterized in that each microchannel of described sample applicator is positioned at same layer or is positioned at different two layers and above different layers, and the microchannel of different layers all is connected with micro through hole with corresponding injection port.
4. sample applicator according to claim 1, is characterized in that described sample applicator comprises two function surfaces, and upper surface comprises injection port, is used for application of sample; Lower surface comprises micro through hole array, is used for restriction and treats fixed sample and substrate contact reaction zone.
5. sample applicator according to claim 1, is characterized in that described sample applicator lower surface is flat surface except the micro through hole zone, and the lower surface area is equal to or less than and treats the point sample chip area.
6. the using method of sample applicator according to claim 1 is characterized in that comprising following steps:
A) sample applicator is placed in vacuum tank and carries out degassed processing at least 40 minutes;
B) sample applicator after degassed processing is aimed at laminating with treating the point sample substrate, wherein sample applicator comprises the one side contact substrate of micro through hole array;
C) drip and treat fixed sample solution at each injection port, make each microchannel form closed system, utilize the degassed silicone rubber block to absorb the microchannel Air and form negative pressure, drive fluid sample and be full of microchannel and micro through hole array;
D) after standing at least 30 minutes, peel off sample applicator from substrate, and substrate is immersed in cleaning fluid and cleans, dry up, complete the micro-array chip preparation.
7. method according to claim 6, is characterized in that a) sample applicator after degassed processing or take out directly application or salable standby at any time in vacuum bag from vacuum tank of step.
8. the application of according to claim 1-5 described sample applicators of any one, is characterized in that short run and DIY preparation for the common lab micro-array chip.
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| CN113189357A (en) * | 2021-05-06 | 2021-07-30 | 中国科学院上海微系统与信息技术研究所 | Preparation method of dipping type sample applicator and micro-cantilever sensor chip |
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